Our goal is to understand the mechanisms of cell fate specification and cellular reorganization that transform the fetal gonad into a functional testis. The first step of testis organogenesis is the commitment of approximately half of the somatic cells in the gonad primordium to the Sertoli cell fate. Sertoli cells surround germ cells and undergo the process of de novo cord formation. This basic process of cell sorting defines two compartments of the testis: the testis cords and the interstitium. The testis cords will become the seminiferous tubules, where sperm are produced in adult life, whereas the androgen-producing Leydig cells and other less well-characterized populations differentiate in the interstitium. Although the process of cord formation is essential for masculinization of the fetus and fertility of the adult, it is not well understood. It has been viewed as a Sertoli-cell-driven process;however, several lines of evidence suggest that interstitial cells play a critical role in shaping the process of testis cord formation. One gene that is essential for cell sorting during gonad morphogenesis in Drosophila is traffic jam (tj). tj encodes a member of the Maf transcription factor family, and promotes organogenesis of the Drosophila gonad via the regulation of cell adhesion molecules (CAMs). The mammalian ortholog of tj, MafB, is expressed in an uncharacterized population of progenitor cells that sort out from Sertoli cells at the beginning of fetal testis formation and give rise to the interstitium of the testis. MafB expression is closely associated with the vasculature, and later becomes restricted to fetal Leydig cells. We hypothesize that MafB plays a role in specification of the cell types of the interstitium and in the formation of testis cords, likely acting through control of CAMs or extracellular matrix (ECM) proteins. We have recently shown that Notch signaling is required for regulation of the progenitor population in the interstitium and for Leydig cell differentiation. In Specific Aim 1, we will investigate the hypothesis that Notch signaling is involved in the specification of MafB-expressing cells, using in vivo and ex vivo Notch gain- and Ibss-of-function analysis via immunocytochemistry, in situ hybridization, and quantitative PCR. In Specific Aim 2, we propose to elucidate the role of MafB-expressing cells during testis morphogenesis, employing PCR, microarray techniques, and genetic methods to determine which CAMs are regulated by MafB. In both aims, we will utilize several transgenic mouse lines for live imaging and fluorescence-activated cell sorting to study testis morphogenesis at the cellular level. This study of cell fate specification and cell-cell interactions will contribute to our basic understanding of organogenesis and provide insight into the developmental basis for infertility and reproductive disorders.